confirmation of conjugation cBSA-Estradiol... How?! - (Mar/25/2007 )

how can i confirm the conjugation of cBSA( Cationized BSA) and Estradiol

-nish-

QUOTE (nish @ Mar 25 2007, 10:16 PM)

how can i confirm the conjugation of cBSA( Cationized BSA) and Estradiol

After desalting on Sephadex G25 ( to remove non conjugated estardiol ) Mass spectrometry will help you to detect ( molecular ion detection). Quantative analisis will help you to detect ratio of CBSA and estradiol in your conjugate. In addition Also may be specific in vitro activity test for activity , as controls - cBSA and estradiol

-circlepoint-

thanks for the replymass spec is beyond my financia;l credit . i require a cost effective method.. can you suggest a method!!

-nish-

QUOTE (nish @ Mar 26 2007, 07:48 PM)

thanks for the replymass spec is beyond my financia;l credit . i require a cost effective method.. can you suggest a method!!

Well. Try to make conjugate cBSA with phenol red dye ( cheap one). This substance contain active hydrogens in ortho to the hydroxyl group on the phenol ring. (which participate in the conjugation ) very similar with estradiol active site. You conjugate will be colored and so visual after sephadex G25 desalting. When you optimize conditions with this model dye. You can make conjugate with estradiol and inject in mice i.p. using alum as adjuvant. As I undersatand you prepare this conjugate to improve antibody production against estadiol.

-circlepoint-

thanks .i have already tried out that method ....i have a clear visual upon loading onto the sephedex G25 upon collection of sample i observed no clear colour.....i even gave a nanodrop reading which showed only one peak which was for cBSA and the other for phenol red .......i will try and standardize thanks again

-nish-

QUOTE (nish @ Mar 28 2007, 07:26 PM)

thanks .i have already tried out that method ....i have a clear visual upon loading onto the sephedex G25 upon collection of sample i observed no clear colour.....i even gave a nanodrop reading which showed only one peak which was for cBSA and the other for phenol red .......i will try and standardize thanks again

Hello Nish!Try to do conjugation according the following protocol:

Dissolve cBSA (10mg\ml) in 200 ul coupling buffer ( 0,1M MES 0,15M NaCl, pH4.7). Ph optimal for Mannich condensation. Mix with 200ul of 10mg\ml estradiol (dissolved in absolute ethanol). Add 50ul additional absolute ethanol ( to increase solubity of estradiol after mixing). Add 50ul 37% formaldehyde(Sigma). Try to make you conjugation at various temperature ( 37 and 57C) in water bath. for 6-24 hour. Separate your Conjugate on Sephadex G25. Try to make it first with phenol red to to see color product. Note! For phenol red - dissolve it in coupling buffer because it water soluble. and so no need to add additional 50ul ethanol.